MECHANISM-BASED INACTIVATION OF HUMAM

A Thesis by

Yi Li

B. S., Jilin University, 2005

Submitted to the Department of Chemistry and the faculty and Graduate School of Wichita State University in partial fulfillment of the requirements for the degree of Master of Science

December 2008

© Copyright 2008 by Yi Li,

All Rights Reserved

ii MECHANISM-BASED INACTIVATION OF HUMAM NEUTROPHIL ELASTASE

I have examined the final copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirement for the degree of Master of Science with a major in Chemistry

______William C. Groutas, Committee Chair

We have read this thesis and recommend its acceptance:

______Erach R. Talaty, Committee Member

______Kandatege Wimalasena, Committee Member

______Lop-Hing, Ho, Committee Member

iii ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Groutas for his kindness, guidance and support throughout my studies and research at Wichita State University, and for the opportunity given to me to broaden my knowledge and interest in organic chemistry and medicinal chemistry. I am very grateful for such a wonderful teacher in my life.

I would also like to thank Dr. Talaty, Dr. Wimalasena and Dr. Ho for serving on my thesis committee. Furthermore, I would like to thank Dr. Alliston for his help with the biochemical studies. I am also thankful to members of my group and all the faculty and staff of the Department of Chemistry at Wichita State University.

Many thanks must be extended to my family and friends for their love and support.

iv ABSTRACT

Chronic obstructive pulmonary disease (COPD) is a major health problem that affects

16 million people in the US, and is currently the fourth most common cause of death.

Although the pathogenesis of COPD is poorly understood, current studies indicate that

COPD is a multi-factorial disorder characterized by a cigarette smoke-induced cycle of oxidative stress, alveolar septal cell apoptosis, a /antiprotease imbalance, and chronic inflammation.

An array of (HNE, PR3), cysteine ( S), and metallo- (MMP-1, MMP-9,

MMP-12) released by , macrophages, and T lymphocytes contribute to the degradation of lung connective tissue and mediate a multitude of signaling pathways associated with the pathophysiology of the disorder. Re-establishment of a protease/antiprotease balance by utilizing potent and selective protease inhibitors is a promising approach for the development of potential therapeutics for COPD.

We describe herein the design, synthesis and biochemical evaluation of a novel class of mechanism-based inhibitors of HNE that exploit the catalytic machinery of the target to generate a Michael acceptor. Subsequent reaction with an nucleophilic residue leads to inactivation of the enzyme. A noteworthy feature of the inhibitors is their ability to interact with the S 1-Sn’ subsites of the target enzyme.

v TABLE CONTENTS

Chapter Page

1. INTRODUCTION ...... 1

1.1 An Overview of COPD...... 1 1.1.1 COPD...... 1 1.1.2 The Cause of COPD...... 1 1.1.3 The Symptoms of COPD ...... 2 1.1.4. The Pathophysiology of Emphysema ...... 2

1.2 An Overview of COPD-Relevant Proteases ...... 3 1.2.1 Proteases ...... 3 1.2.1.1 Serine Proteases ...... 3 1.2.1.2 Matrix Metalloproteases ...... 5 1.2.1.3 Cysteine proteases...... 5 1.2.2 Elastin ...... 5 1.2.3 Antiproteinases ...... 6 1.2.4 The Imbalance of Proteases and Antiproteases ...... 6

1.3 Inhibition...... 7 1.3.1 Substrate Specificity ...... 7 1.3.2 Mechanism of Action...... 10 1.3.3 Inhibition...... 11 1.3.3.1 Irreversible inhibitors...... 11 1.3.3.2 Reversible inhibitors ...... 14

2. DESIGN RATIONALE AND RESEARCH GOALS ...... 17

2.1 Inhibitor Design Rationale...... 17

2.2 Research Goals...... 21

3. EXPERIMENTAL...... 22

3.1 General...... 22

3.2 Synthetic procedures...... 22

3.3 Biochemical Assays...... 35

4. RESULTS AND DISSCUSION...... 37

4.1 Synthesis ...... 37

vi TABLE CONTENTS (continued)

Chapter Page

4.2 Biochemical Results...... 39

5. CONCLUSIONS...... 41

REFERENCES ...... 42

vii

LIST OF TALBES

Table Page

Table 1 Substrate Specifity of Used in the Proposed Research...... 8

Table 2 Physical and Spectra Data of Compounds 1-9...... 32

Table 2 Physical and Spectra Data of Compounds 1-9 (continued) ...... 33

Table 2 Physical and Spectra Data of Compounds 1-9 (continued) ...... 34

Table 3 Biochemical Evaluation of Inhibitors (I) ………………………………………39

viii

LIST OF FIGURES

Figure Page

Figure 1 Berger and Schechter Nomenclature for Proteases ...... 7

Figure 2 The Reaction Mechanism ...... 11

Figure 3 Kinetic Scheme for Mechanism-Based Inhibitors...... 12

Figure 4 Competitive Inhibition...... 14

Figure 5 Kinetic Scheme of Reversible Inhibitors...... 15

Figure 6 Human Neutrophil Elastase/Turkey Ovomucoid Inhibitor Complex...... 17

Figure 7 Network of Hydrogen Bonds and Hydrophobic Binding Interactions...... 18

Figure 8 Structure-Based Design of 1, 2, 5-thiadiazolidin-3-one 1, 1-dioxide Scaffold . 19

Figure 9 3-Aza Grob Fragmentation Reaction...... 20

Figure 10 Proposed Mechanism of Inhibition...... 21

Figure 11 Structures of Compounds 1-9...... 28

Figure 11 Structures of Compounds 1-9 (continued)...... 29

Figure 11 Structures of Compounds 1-9 (continued)...... 30

Figure 11 Structures of Compounds 1-9 (continued)...... 31

Figure 12 Time-Dependent Loss of Enzymatic Activity...... 40

Figure 13 Progress Curves of Inhibitor 8b with HNE ...... 40

ix

LIST OF SCHEMES

Scheme Page

Scheme 1 Synthesis of Inhibitors (I) ...... 38

x

LIST OF ABBREVIATIONS AND TERMS

COPD Chronic Obstructive Pulmonary Diseases

NMR Nuclear Magnetic Resonance

TLC Thin Layer Chromatography

ClSO 2NCO Chlorosulfonyl isocyanate t-BuOH tertiary-Butyl alcohol

TEA Triethylamine

DCM Dichloromethane

EtOAc Ethyl Acetate

NaH Sodium hydride

THF Tetrahydrofuran

TFA Trifluoroacetic acid

DMF Dimethylformamide

CH 3CN Acetonitrile

NaBH 4 Sodium borohydride

(CH 3)3SiI Trimethyl silyliodide

NaCl Sodium chloride

HCl Hydrogen chloride

NaHCO 3 Sodium bicarbonate

xi CHAPTER 1 INTRODUCTION

1.1 An Overview of COPD

1.1.1 COPD

Chronic Obstructive Pulmonary Disease (COPD) is the fourth common cause of death in US. It is a multifactorial inflammatory disorder characterized by enlargement of the air spaces and airflow obstruction due to chronic bronchitis and pulmonary emphysema.

COPD affects more than 16 million Americans and is the fourth most common cause of death in the U.S. It is the only major disease that is continuing to increase in both prevalence and mortality. The cost of care of COPD patients is estimated to be ~$24 billion/yr. 1 COPD is associated with influx of macrophages, neutrophils and T lymphocytes to the lungs, presence of pro-inflammatory mediaters, oxidative stress, apoptosis, extracellular release of an array of proteases and chronic inflammation.

1.1.2 The Cause of COPD

Smoking is unequivocally the primary cause of COPD, which is why it is considered to be a preventable disease. This conclusion is justified by the fact that, at least in the United

States, the condition is seen so uncommonly among nonsmokers. Although the most critical factor for acquiring COPD is cigarette smoking, only 15 to 20% of chronic smokers get this disease. Several epidemiologic studies 2-5 have suggested familial clustering of the disease. This suggests that genetic factors are likely to have a role in determining an individual’s susceptibility to COPD.

1 1.1.3 The Symptoms of COPD

In emphysema, the walls of the air sacs are damaged and individual air sacs collapse into fewer, larger air sacs, which lose their ability to transfer gases in and out of the bloodstream. In addition, air is trapped in these large, diseased, nonfunctional air sacs.

Over time, this causes the air sacs, and the lungs, to become bigger (hyperinflate), especially with exercise. The hyperinflation is accompanied by increased difficulty breathing and shortness of breath. In chronic bronchitis, excessive amounts of mucus are produced. This mucus is often of poor quality-tenacious and difficult to expel, even with coughing. This mucus, which serves as a defense against bacteria, viruses, and other foreign particles, is seriously compromised. Particles remaining in the lungs cause infection and inflammation. It then becomes increasingly difficult for the already damaged cilia to sweep the particle-laden mucus out of the lungs. The retained mucus further narrows the airways, and this narrowing acts in concert with hyperinflation to make it even more difficult to breathe. 6

1.1.4. The Pathophysiology of Emphysema

Emphysema is defined as the enlargement of peripheral air spaces of the lung, including respiratory bronchioles, alveolar ducts, and alveoli, accompanied by destruction of the walls of these structures.7-8 Inherited deficiency of a1-antitrypsin, which is the primary inhibitor of neutrophil elastase, predisposes individuals to early onset emphysema 9 and intrapulmonary instillation of elastolytic enzymes in experimental animals causes emphysema 10 .Together, these findings have led to the hypothesis that emphysema results from proteolytic injury directed especially against elastin, the main component of elastic fibers.

2 1.2 An Overview of COPD-Relevant Proteases

1.2.1 Proteases

Proteases or proteolytic enzymes form one of the largest and most important groups of enzymes. Proteases selectively catalyze the hydrolysis of peptide bonds and can be divided into five major classes: aspartic, serine, cysteine, metallo and threonine proteases.

Proteases are involved in numerous important physiological processes including protein turnover, digestion, blood and wound healing, fertilization, cell differentiation and growth, cell signaling, the immune response, and apoptosis.

Uncontrolled or undesired proteolysis can lead to many disease states including emphysema, stroke, viral infections, cancer, Alzheimer’s disease, inflammation, and arthritis.11-12 Among the proteases, serine proteinases including Human Neutrophil

Elastase (HNE), (PR3), (Cat G), Metalloproteases (MMP)

(MMP-12, MMP-9) and Cysteine proteases (Cathepsin S) have been implicated in COPD.

1.2.1.1 Serine Proteases

Serine proteases or serine are a class of peptidases (enzymes that cleave peptide bonds in proteins) that are characterized by the presence of a serine residue in the active site of the enzyme. Serine proteases participate in a wide range of functions in the body, including blood clotting, immunity, and inflammation, as well as contributing to digestive enzymes in both prokaryotes and eukaryotes.

The three serine proteases of the -like clan that have been studied in greatest detail are chymotrypsin, , and . All three enzymes are synthesized by the pancreatic acinar cells, secreted in the small intestine and are responsible for catalyzing the hydrolysis of peptide bonds. All three of these enzymes are

3 similar in structure, as shown through their X-ray structures. The differing aspect lies in the peptide bond which is being cleaved, which is called the scissile bond. The different enzymes, like most enzymes, are highly specific in the reactions they catalyze. Each of these digestive serine proteases targets different regions of a polypeptide chain, based upon the side chains of the residues surrounding the site of cleavage:

(a) Chymotrypsin is responsible for cleaving peptide bonds following an aromatic amino acid residue. Preferred residues include phenylalanine, tryptophan and tyrosine, which fit snugly into a hydrophobic pocket.

(b) Trypsin is responsible for cleaving peptide bonds following a positively-charged amino acid residue. Instead of having the hydrophobic pocket of the chymotrypsin, there exists an residue at the base of the pocket. This can then interact with positively-charged residues such as arginine and lysine on the substrate peptide to be cleaved. The pocket that is in "trypsin" and "chymotrypsin" is now partially filled with valine and threonine, rendering it a mere depression, which can accommodate these smaller amino acid residues.

(c) Elastase breaks down elastin, an elastic fiber that, together with collagen, determines the mechanical properties of connective tissue. The neutrophil form breaks down the outer membrane protein A (OmpA) of E. coli and other Gram-negative bacteria, and also breaks down Shigella virulence factors. This is accomplished through the cleavage of peptide bonds in the target proteins. The specific peptide bonds cleaved are those on the carboxy side of small, hydrophobic amino acids such as glycine, alanine, and valine.

4 1.2.1.2 Matrix Metalloproteases

MMPs are a family of zinc endopeptidases that are structurally and functionally related.

The MMP family can be classified into five major groups based on the substrates: (1) collagenases (MMP-1, -8, -13), (2) gelatinases (MMP-2, -9), (3) stromelysins (MMP-3, -

10, -11), (4) a heterogeneous subgroup including matrilysin (MMP-7), enamelysin

(MMP-20), macrophage metalloelastase (MMP-12), and MMP-19, and (5) membrane- type MMPs (MMP-14 to MMP-17 and -24, -25 or MT1–6-MMP). The MMPs play an important role in tissue remodeling associated with various physiological and pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis and metastasis. MMP-2 and MMP-9 are thought to be important in metastasis.

MMP-1 is thought to be important in rheumatoid and osteo-arthritis. Evidence suggests that matrix metalloproteases (MMPs) have the ability to bring about hydrolysis of connective tissue matrix proteins. 13

1.2.1.3 Cysteine proteases

Cysteine proteases have a common catalytic mechanism that involves a nucleophilic cysteine thiol in a . The role of cysteine porteinases in COPD has not been defined, although they do contribute to the elastolytic activity of alveolar macrophages in

COPD patients. 14

1.2.2 Elastin

Poor regulation of the activity of the released proteases results in the degradation of lung elastin (hydrophobic component of lung connective tissue) and other components of the extracellular matrix. Elastin is a protein in connective tissue that is elastic and allows

5 many tissues in the body to resume their shape after stretching or contracting. It is an important load-bearing tissue in the bodies of mammals and used in places where mechanical energy is required to be stored. Elastin serves an important function in arteries and is particularly abundant in large elastic blood vessels such as the aorta. It is also very important in the lungs, elastic ligaments, the skin, the bladder, elastic cartilage, and the intervertebral disc above the sacroiliac.15

1.2.3 Antiproteinases

Antiproteinases are endogenous proteins that inhibit proteolytic enzymes. Endogenous antiproteinases are natural protease inhibitors, including the family of lipocalin proteins, which play a role in cell regulation and differentiation. Lipophilic ligands, attached to lipocalin proteins, have been found to possess tumor protease inhibiting properties, such as alpha 1-antitrypsin (A1AT), secretory leukocyte proteinase inhibitor (SLPI), monocyte/neutrophil elastase inhibitors (MNEI), tissue inhibitors of metalloprotreases

(TIMPs) and cystatins. Examples of protease inhibitors are the class of (serine protease or peptidase inhibitors), incorporating alpha 1-antitrypsin. Other serpins are complement 1-inhibitor, , alpha 1-antichymotrypsin, inhibitor 1 (coagulation, ) and the recently discovered neuroserpin.

1.2.4 The Imbalance of Proteases and Antiproteases

Smoking causes an imbalance between proteinases that digest elastin (and other structural proteins) and antiproteinases that protect against this. The pathophysiology observed in COPD arises from an imbalance between the levels of proteases and their endogenous protein inhibitors. This suggests that either inhibiting these proteolytic

6 enzymes or increasing endogenous antiproteinases may be beneficial and theoretically should prevent the progression of airflow obstruction in COPD. Considerable progress has been made in identifying the enzymes involved in elastolytic activity in emphysema and in characterizing the endogenous antiproteinases that counteract this activity. The fact that there are so many proteinases implicated in COPD might mean that blocking a single enzyme may not have a complete effect. 16

1.3 Inhibition

1.3.1 Substrate Specificity

Figure 1 Berger and Schechter Nomenclature for Proteases

The Berger and Schechter nomenclature has become generally accepted and used to describe the interaction of a substrate with a protease (Figure 1). The system is based on a schematic interaction of amino acid residues of the substrate with specific binding subsites located at the active site of a protease. By convention, the subsites on the protease are designated as S (for subsites) and the amino acid residues of substrate are designated as P (for amino acid residue in peptides). The number of each residue is given from the scissile bond (the bond which is cleaved in the enzymatic reaction). S1, S 2,

7 S3,…Sn and S1′, S 2′, S 3′,…Sn′ correspond to the enzyme subsites on either side of the scissile bond. Each subsite accommodates a corresponding amino acid residue side chain designated P 1, P 2, P 3,… Pn and P 1′, P 2′, P 3′,…Pn′ of the substrate or (inhibitor). S 1 is the primary substrate specificity subsite, and the amide bond between P1 and P1′ is the scissile bond. 17

Substrate Specificity of Human Neutrophil Elastase, Proteinase 3 and Cathepsin

G. In order to achieve the specific aims of this thesis, we have chosen to focus initially on the COPD-relevant proteases PR3, HLE, Cat G, and MMP-12. The overriding criterion used in the selection of these enzymes is their clinical relevance with respect to COPD. A brief overview of three of the target enzymes (HLE, PR 3, Cat G) and the rationale underlying the design and synthesis of the various inhibitors are given below.

Table 1 Substrate Specifity of Enzymes Used in the Proposed Research Enzyme Substrate Specificity a

– S 4 – S 3 – S 2 – S 1 – S 1’ – S 2’ – S 3’ – S 4’ –

PR3 Ala Ala Pro Abu b Lys Gly Asp -

HNE Ala Val Pro Val c - - - -

Cat G Ala Val Pro Phe - - - - a b the S n’ subsites of HLE and Cat G have not been mapped; Norval works equally well; cLeu works equally well

It was mentioned earlier that COPD involves the interplay of a range of proteolytic enzymes, including PR 3 and HNE. PR 3 and HNE have the ability to degrade lung elastin, the major component of lung connective tissue, and basement membrane components, as well as activate various metalloproteases. PR 3 hydrolyzes elastin with the same avidity as HNE and induces emphysema in hamsters commensurate with that of

8 HNE. Furthermore, SLPI (an endogenous protein that protects the upper airways from proteolysis) regulates the activity of HNE but does not inhibit PR 3 and is, in fact, degraded by the enzyme. Despite the apparent prominent role that PR 3 plays in COPD, it has received scant attention compared to HNE. Ascertaining the specific role played by

PR 3 and HNE in COPD requires the availability of highly selective inhibitors of each enzyme.

The primary structures of PR 3, HNE and Cat G are known and their have been cloned. HNE is a basic, 218 amino acid single polypeptide glycoprotein (Mr 29,500) whose primary structure shows considerable homology with PR 3 (54%), and Cat G

(37%). PR 3 is homologous with Cat G (35%) and α-chymotrypsin (30%). X-ray crystal structures of HNE, PR 3, and Cat G complexed to low molecular weight inhibitors have been reported. These enzymes have extended binding sites and prefer hydrophobic substrates/inhibitors. While the S 1-S4 subsites of PR 3, HNE and Cat G have been mapped using peptidyl chromogenic substrates (Table 1), only the S’ subsites of PR 3 have been mapped. It should be noted that because PR 3, HLE and Cat G show a preference for the same P 2-P4 residues, the primary focus of our studies is on the exploitation of differences in their primary specificity (S 1) and S’ subsites.

As shown in Table 1, the primary specificity pocket (S 1) of PR 3, HNE and Cat G increases in size in the order PR 3

HNE to Ile 190 and Asp 213 in PR 3 respectively, which reduce the size of the S 1 pocket.

HNE prefers medium size P 1 alkyl groups (isopropyl, isobutyl, n-butyl), while the larger

S1 pocket of Cat G shows a strong preference for a P 1 Phe residue. While inhibitors have

9 been designed that inhibit only Cat G and are devoid of any inhibitory activity toward PR

3 and HNE, or vice versa, efforts to design inhibitors that inhibit PR 3 with high selectivity over HLE have only been partially successful thus far, gravely hampering efforts to delineate the precise role PR 3 and HNE play in COPD. The notion is advanced that the structure-based design of suitably-embellished inhibitors, such as (I), that exploit the subtle differences that exist in the S’ subsites of the target enzymes may lead to highly selective inhibitors of these enzymes.

1.3.2 Mechanism of Action

The main player in the catalytic mechanism in the chymotrypsin and clan enzymes mentioned above is the catalytic triad. The triad is located in the active site of the enzyme, where catalysis occurs, and is preserved in all serine protease enzymes. The triad is a coordinated structure consisting of three essential amino acids: (His

57), serine (Ser 195) and aspartic acid (Asp 102). Located very near one another near the heart of the enzyme, these three key amino acids each play an essential role in the cleaving ability of the proteases. The mechanism of action of mammalian serine proteases (HNE, PR3, Cat G) is similar to that shown in Figure 2.

In the event of catalysis, an ordered mechanism occurs in which several intermediates are generated (Figure 2). The catalysis of the peptide cleavage can be seen as a ping-pong catalysis, in which a substrate binds (in this case, the polypeptide being cleaved), a product is released (the N-terminus "half" of the peptide), another substrate binds (in this case, water), and another product is released (the C-terminus "half" of the peptide).

10

Figure 2 The Serine Protease Reaction Mechanism

1.3.3 Inhibition

1.3.3.1 Irreversible inhibitors

Irreversible inhibitors usually covalently modify an enzyme, therefore inhibition cannot therefore be reversed. Irreversible inhibitors often contain reactive functional groups such as nitrogen mustards, isocyanates, α-chloromethyl ketones, etc. These electrophilic groups react with amino acid side chains to form covalent adducts. The residues modified are those with side chains containing nucleophiles such as hydroxyl or

11 sulfhydryl groups; these include the amino acids serine, cysteine, threonine or tyrosine.18

Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein structure but by specifically altering the active site of their target. For example, extremes of pH or temperature usually cause denaturation of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentrated hydrochloric acid will hydrolyze the peptide bonds holding proteins together, releasing free amino acids.19

Mechanism-based (suicide) inhibitors are one type of irreversible inhibitor. They have been extensively used in mechanistic enzymology, and drug design and discovery. 20

Indeed, several drugs currently in the clinic exert their effects by utilizing a mechanism- based type of inhibition. A mechanism-based inhibitor is typically a molecule of low reactivity that acts as a substrate and is processed by the catalytic machinery of an enzyme, generating a reactive electrophilic species (for example, an acid chloride, ketene, isocyanate or a Michael acceptor) that remains tethered to the active site. Subsequent reaction with an active site nucleophilic residue leads to irreversible inactivation of the enzyme since only the target enzyme can engage the inhibitor and optimal selectivity is likely to be attained. Furthermore, while in principle other enzymes that share the same catalytic mechanism as the target enzyme can also be inhibited, this obstacle can be circumvented by incorporating into the inhibitor recognition elements that exploit differences in the S and S’ subsites of these enzymes.

Figure 3 Kinetic Scheme for Mechanism-Based Inhibitors

12

As shown in the figure 3, mechanism-based inhibitors form a reversible non-covalent complex with the enzyme (EI) which is transformed into a modified "dead-end complex"

EI*, ultimately leading to E-I*. The rate at which E-I* is formed is called the inactivation rate or k inact .

The binding and inactivation steps of this reaction are investigated by incubating the enzyme with inhibitor and assaying the amount of activity remaining over time. The activity will be decreased in a time-dependent manner, usually following exponential decay. Fitting these data to a rate equation gives the rate of inactivation at this concentration of inhibitor. This is done at several different concentrations of inhibitor. If a reversible EI complex is involved the inactivation rate will be saturable and fitting this

21 curve will give k inact and K I.

Another method that is widely used in these analyses is mass spectrometry. Here, accurate measurement of the mass of the unmodified native enzyme and the inactivated enzyme gives the increase in mass caused by reaction with the inhibitor and shows the stoichiometry of the reaction.22 This is usually done using a MALDI-TOF mass spectrometer. In a complementary technique, peptide mass fingerprinting involves digestion of the native and modified protein with a protease such as trypsin. This will produce a set of peptides that can be analyzed using a mass spectrometer. The peptide that changes in mass after reaction with the inhibitor will be the one that contains the site of modification.

13 1.3.3.2 Reversible inhibitors

Reversible inhibitors bind to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis.

There are three kinds of reversible enzyme inhibitors. They are classified according to the effect of varying the concentration of the enzyme's substrate on the inhibitor.23

In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in the figure on the left. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate, i.e., by out- competing the inhibitor. Competitive inhibitors are often similar in structure to the real substrate (Figure 4).

Figure 4 Competitive Inhibition

14 As in Figure 4, substrate (S) and inhibitor (I) compete for the active site. In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the enzyme's substrate.

However, the binding of the inhibitor affects the binding of the substrate, and vice versa.

This type of inhibition can be reduced, but not overcome by increasing concentrations of substrate. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.

Non-competitive inhibition is a form of mixed inhibition where the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration of the inhibitor.

Figure 5 Kinetic Scheme of Reversible Inhibitors

Reversible inhibition can be described quantitatively in terms of the inhibitor's binding to the enzyme and to the enzyme–substrate complex, and its effects on the kinetic constants of the enzyme. In the classic Michaelis–Menten scheme (Figure 5), an enzyme

(E) binds to its substrate (S) to form the enzyme–substrate complex ES. Upon catalysis, this complex breaks down to release product P and free enzyme. The inhibitor (I) can bind to either E or ES with the dissociation constants K i or K i', respectively. Competitive inhibitors can bind to E, but not to ES. Competitive inhibition increases K m (i.e., the inhibitor interferes with substrate binding), but does not affect V max (the inhibitor does not hamper catalysis in ES because it cannot bind to ES). Non-competitive inhibitors

15 have identical affinities for E and ES (K i = K i'). Non-competitive inhibition does not change K m (i.e., it does not affect substrate binding) but decreases Vmax (i.e., inhibitor binding hampers catalysis).

Mixed-type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (K i ≠ K i'). Thus, mixed-type inhibitors interfere with substrate binding (increase K m) and hamper catalysis in the ES complex (decrease V max ).

When an enzyme has multiple substrates, inhibitors can show different types of inhibition depending on which substrate is considered. This results from the active site containing two different binding sites within the active site, one for each substrate. For example, an inhibitor might compete with substrate A for the first , but be a non- competitive inhibitor with respect to substrate B in the second binding site.24

16 CHAPTER 2 DESIGN RATIONALE AND RESEARCH GOALS

2.1 Inhibitor Design Rationale

In designing scaffolds I, the X-ray crystal structure of the HNE-TOMI complex 25

(PDB 1HNL) was utilized. In this complex, the protein inhibitor occupies an extended binding region stretching from S 5 to S 3’, corresponding to residues -Pro-Ala-Cys-Thr-

Leu-Glu-Tyr-Arg- of the inhibitor (Figure 6).

Figure 6 Human Neutrophil Elastase/Turkey Ovomucoid Inhibitor Complex

The P 3-P1 residues (-Cys-Thr-Leu-) of TOMI form an antiparallel β-sheet binding arrangement with the Ser-214 to Val-216 segment of HNE. Several hydrogen bonds are observed, including a pair of hydrogen bonds between the P 3 residue of the inhibitor and

Val-216 of the enzyme. This is analogous to the network of hydrogen bonds and

17 hydrophobic binding interactions observed between HNE and peptidyl substrates (Figure

7). 26

Figure 7 Network of Hydrogen Bonds and Hydrophobic Binding Interactions

The backbone conformation of the inhibitor recognition loop (residues P 2-P2’) was locked by linking the nitrogen atoms of the P 1 and P 1’ residues with an SO 2 or CH 2 group, thereby generating cyclic template (Figure 8).

18

Figure 8 Structure-Based Design of 1, 2, 5-thiadiazolidin-3-one 1, 1-dioxide Scaffold

It was anticipated that the conversion of the flexible peptide inhibitor into rigid cyclic structure would provide an entropic advantage that would more than compensate for the loss of one of the hydrogen bonds between HLE and TOMI. Importantly, sufficient flexibility is embodied in template (I) so that, in addition to diversity elements R 2 and R 3, the primary specificity residue P1 was intended to serve both as a sensitive probe of the

S1 subsite and as a third diversity element (vide infra).

We have earlier described classes of mechanism-based inhibitors of HNE, that inactivate the enzyme via an enzyme-induced Lossen 27 or Gabriel Colman 28 rearrangement. More recently, we have reported two novel classes of mechanism-based inhibitors of HNE that inactivate the enzyme by generating a Michael acceptor via a sulfonamide fragmentation process.29 The biochemical rationale underlying the design of the first example of a mechanism-based inhibitor of HNE that generates a Michael acceptor via a 3-aza Grob fragmentation process (Figure 9)30 is described in this thesis.

19

Figure 9 3-Aza Grob Fragmentation Reaction

The design of inhibitor (I) was based on the following considerations: (a) chemical system having a nucleophilic atom with a negative charge or unshared electron pair and a leaving group in a 1, 4-relationship are known to undergo a facile heterolytic fragmentation; (b) literature reports describing examples of amide bond cleavage that occur via a 3-aza Grob fragmentation process and, (c) a heterocyclic scaffold (1, 2, 5- thiadiazolidin-3-one 1,1 dioxide) known to bind to the active site of HNE and related

(chymo)trypsin-like serine proteases in a substrate-like fashion with appended recognition elements that interact with the S and S’ subsites was used to assemble inhibitor (I) (Figure 10). It was envisaged that compound (I) would inactivate HNE via a sequence of steps involving the formation of a tetrahedral intermediate that undergoes a facile 3-aza Grob fragmentation to yield a Micheal acceptor that either reacts with a nearby nucleophilic residue (His57) or a water-mediated process to generate a stable acyl enzyme, leading to inactivation of the enzyme (Figure 10).

20

Figure 10 Proposed Mechanism of Inhibition

2.2 Research Goals

Based on the forgoing discussion, the research goals were the following:

(a) Structure-based design and synthesis of novel mechanism-based inhibitors for HNE that interact with the S-Sn’ subsites and inactivate the enzyme via a 3-Aza Grob fragmentation reaction and,

(b) Biochemical studies aimed at determining the inhibitory potency, enzyme selectivity, mechanism of action, and stability of the inhibitors.

21 CHAPTER 3

EXPERIMENTAL

3.1 General

Melting points were recorded on a Mel-Temp apparatus and are uncorrected. 1H NMR spectra of the synthesized compounds were recorded on Varian XL-300 or XL-400 spectrometers. Human neutrophil elastase was purchased from Elastin Products Co.,

Owensville, MO. Methoxysuccinyl Ala-Ala-Pro-Val p-nitroanilide was purchased from

Sigma Chemicals Co., St Louis, MO. Aldrich 230-400 mesh silica gel was used for flash chromatography. Representative detailed synthetic procedures are described below.

3.2 Synthetic procedures

Synthesis of compound 1a

Thionyl chloride (27.87 g; 234 mmol) was added dropwise to methanol (85 mL) kept in an ice-salt bath. (L)H-Nva-OH (25.00g; 213 mmol) was then added in small portions.

The ice-salt bath was removed and the reaction mixture was heated to about 40 ℃ using water bath. The reaction mixture was stirred for 2 hours at 40 ℃. The solvent was removed and the residue was collected by suction filtration and washed with ether (100 mL). The solid was dried in a desiccator to give 1a (34.17 g; 96%). The crude product was used in run the next reaction without purification.

Synthesis of compound 2a

A solution of N-chlorosulfonyl isocyanate (28.73 g; 203 mmol) in dry methylene chloride (140 mL) was cooled in an ice-water bath and a solution of t-butyl alcohol

(15.05 g; 203 mmol) in dry methylene chloride (60 mL) was added dropwise with stirring.

22 The mixture was stirred for another 15 minutes at 0℃ and the resulting mixture was added dropwise to a solution of 1a (34.17 g; 203 mmol) in dry methylene chloride (200 mL) and TEA (1.08 g; 406 mmol) kept in an ice-water bath. The ice-water bath was removed and the mixture was stirred at room temperature overnight. The reaction mixture was washed with 5% aqueous HCl (2 x 150 mL) and brine (150 mL). The organic layer was dried over anhydrous sodium sulfate and the solvent was removed to give a crude product 2a (82.23 g; 100%) as a white solid. The crude product was used in the next step without purification.

Compound 2b was synthesized using a similar procedure.

Synthesis of compound 3a

Compound 2a (62.99 g; 203 mmol) was dissolved in TFA (200 mL). The reaction mixture was stirred at room temperature overnight. Excess TFA was removed and the residue was taken up by ethyl acetate (800 mL) and washed with saturated NaHCO 3 (5 x

150 mL) and brine (150 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed, leaving a crude product 3a (33.28 g; 78%) as a white solid.

The crude product was used in the next step without purification.

Compound 3b was synthesized using a similar procedure.

Synthesis of compound 4a

A solution of compound 3a (33.28 g; 158 mmol) in dry THF (300 mL) was kept in an ice-water bath and treated portionwise with 60% sodium hydride (9.48 g; 237 mmol) under N 2. The reaction mixture was stirred overnight at room temperature. The solvent was removed and the residue was dissolved in cold water (150 mL). The pH was adjusted to 6-7 with 6 M HCl. The starting material was extracted with ethyl acetate (150 mL) and

23 the aqueous layer was separated and acidified with 6 M HCl to pH 1. The product was extracted with ethyl acetate (3 x 150 mL) and the combined organic extracts were dried over anhydrous sodium sulfate, and the solvent was removed, leaving a crude product 4a

(28.36 g; 100%) as a yellow oil. The crude product was used in the next step without purification.

Compound 4b was synthesized using a similar procedure.

Synthesis of compound 5a

4-Chlorobenzenethiol (8.7 g; 60 mmol) was dissolved in anhydrous DMF (20 mL).

The solution was treated with TEA (13.7g; 135 mmol), and then stirred for 15 minutes.

Ethyl 5-bromobutyrate (12.55g; 60 mmol) was added and the reaction mixture was stirred overnight. Ethyl acetate (200 mL) was added, and the organic layer was washed with 5% aqueous HCl (50 mL), 5% aqueous NaHCO 3 (50 mL) and brine (50mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed, leaving a crude product which was purified by flash chromatography (silica gel/ethyl acetate/hexane) to give compound 5a (11.57 g; 71%) as a white solid.

Compounds 5c , 5d , 5e and 5f were synthesized using a similar procedure.

Synthesis of compound 5c’

To a solution of compound 5c (45.50 g; 202 mmol) in THF (300 mL) was added TEA

(20.44 g; 202 mmol), followed by di-t-tutyl dicarbonate (61.00 g; 280 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed and ethyl acetate (800 mL) was added and the solution was washed with brine (250 mL),

5% aqueous HCl (2 x 250 mL) and brine (250 mL). The organic layer was dried over anhydrous Na 2SO 4 and the solvent was removed to give a crude product which was

24 purified by flash chromatography (silica gel/ethyl acetate/hexane) to give compound 5c’

(37.25 g; 56%), which was white solid.

Compound 5d’ was synthesized using a similar procedure.

Synthesis of compound 6a

N-chlorosuccinimide (1.05 g; 7.85 mmol) was added in 2 portions over 15 minutes to a solution of compound 5a (2.14 g; 7.85mmol) in benzene (10 mL) and CCl4 (10 mL) and the reaction mixture was stirred at room temperature overnight. The reaction was monitored using KI-starch paper. The precipitated succinimide was filtered off and the solvent was removed to give compound 6a (1.93 g) as a yellow oil. The crude product was used in the next step without purification.

Synthesis of compound 7a

To a solution of compound 4a (19.6 g; 110 mmol) in TEA (11.13 g; 110 mmol) and anhydrous DMF (150 mL) was added compound 6a (30.60 g; 110 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed using vacuum pump 50 ℃/3 hours. The residue was dissolved in ethyl acetate (600 mL) and washed with brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed, leaving a crude product which was purified by flash chromatography (silica gel/ethyl acetate/hexane) to give compound 7a (6.02 g; 13%) as a yellow oil.

Compounds 7b , 7c’ , 7d’ , 7e and 7f were synthesized using a similar procedure.

Synthesis of compound 8a

A solution of compound 7a (22.98 g; 52.83 mmol) in dry acetonitrile (100 mL) was treated with 60% sodium hydride (2.11 g; 52.83 mmol) at 0 ℃. After the solution was

25 stirred for 15 minutes, methyl iodide (18.29 g; 128.85 mmol) was added and the reaction mixture was stirred for 4 hours at room temperature. The solvent was removed and the residue was taken up in methylene chloride (800 mL). The organic layer was washed with water (125 mL). The organic layer was dried over anhydrous sodium sulfate and the solvent was removed, leaving a crude product which was purified by flash chromatography (silica gel/ethyl acetate/hexane) to give compound 8a (7.87 g; 33%) as a yellow oil.

Compounds 8b , 8c’ , 8d’ , 8e and 8f were synthesized using a similar procedure.

Synthesis of compound 8c

A solution of compound 8c’ (2.58 g; 5 mmol) in TFA (20 mL) was stirred over night at room temperature. Excess TFA was removed and the residue was dissolved in ethyl acetate (80 mL) and washed with saturated NaHCO 3 (3 x 20 mL) and brine (20 mL). The organic layer was removed to give a crude product which was purified by flash chromatography (silica gel/ethyl acetate/hexane) to give compound 8c (1.16 g; 59%) as a yellow oil.

Compound 8d was synthesized using a similar procedure.

Synthesis of compound 9a

To a solution of phthalic anhydride (0.37 g; 2.5 mmol) in dry THF (20 mL) was added dropwise a solution of compound 8d (1.04 g; 2.5 mmol) in dry THF (10 mL). The reaction mixture was stirred at room temperature overnight. The solvent was removed and the residue was taken up in ethyl acetate (15 mL). The organic layer was acidified with 15% aqueous HCl solution (15 mL) and the aqueous layer was extracted by ethyl acetate (15 mL). The combined organic layer was washed with brine (15 mL) and dried

26 over anhydrous Na 2SO 4. The solvent was removed to give a crude product which was purified by flash chromatography (silica gel/ethyl acetate/hexane) to give compound 9a

(0.60 g; 42%), which was yellow oil.

Compound 9b was synthesized using a similar procedure.

Synthesis of compound 9c

To a solution of phenethyl isocyanate (0.37 g; 2.5 mmol) in dry CH 2Cl 2 (20 mL) was added a solution of compound 8d (1.04 g; 2.5 mmol) in dry THF (10 mL). The reaction mixture was stirred at room temperature overnight. The solvent was removed and the residue was taken up in ethyl acetate (30 mL). The organic layer was washed with 5% aqueous HCl (15 mL) and brine (15 mL). It was dried over anhydrous Na 2SO 4 and the solvent was removed to give a crude product which was purified by flash chromatography (silica gel/ethyl acetate/hexane) to give compound 9c (0.97 g; 69%) as a light yellow oil.

The structures of compounds 1-9 are listed in Figure 10 and their spectral properties are shown in Table 1.

27

Figure 11 Structures of Compounds 1-9

28

Figure 11 Structures of Compounds 1-9 (continued)

29 Cl NHBoc

O S O S (DL) (DL) O O (L) N (L) N N S HN S O O O O O O 7d' 8b NH NHBoc 2

O S O S (DL) (DL) O O N (L) N (L) N S N S O O O O O 8c O 8c'

NHBoc NH2

O S O S (DL) (DL) O O N N (L) (L) N S N S O O O O O 8d' O 8d Figure 11 Structures of Compounds 1-9 (continued)

30

Figure 11 Structures of Compounds 1-9 (continued)

31 Table 2 Physical and Spectra Data of Compounds 1-9 Compound MF MW 1H NMR Data ( δ) (CDCl3)0.98(t,3H),1.55(m,2H),2.10(m,2H),3.80(s,3H), 1a C H ClNO 167.63 6 14 2 4.20(t,1H),8.68(d,3H)

(CDCl3)0.95(t,3H),1.40(m,2H),1.50(s,9H),1.74(m,2H), 2a C H N O S 310.37 11 22 2 6 3.77(s,3H),4.10(m,1H),6.10(d,1H),8.09(s,1H)

(CDCl3)0.95(t,6H),1.48(s,9H),1.60(m,1H),1.83(m,2H), 2b C H N O S 324.39 12 24 2 6 3.75(s,3H),4.20(m,1H)

(CDCl3)0.95(t,3H),1.42(m,2H),1.70(m,2H),3.79(s,3H), 3a C H N O S 210.25 6 14 2 4 4.10(m,1H),5.10(s,2H),5.78(d,1H)

(CDCl3)0.95(t,6H),1.58(m,1H),1.83(m,2H),3.78(s,3H), 3b C H N O S 224.28 7 16 2 4 4.20(m,1H)

(DMSO)0.95(t,3H),1.38(m,2H),1.82(m,2H),4.18(q,1H), 4a C H N O S 178.21 5 10 2 3 8.23(s,1H)

4b C6H12 N2O3S 192.24 (DMSO)0.95(q,6H),1.58(m,2H),1.70(m,1H),4.18(q,1H)

(CDCl3)1.95(t,2H),2.45(t,2H),2.95(t,2H),3.65(s,3H), 5a C H ClO S 244.73 11 13 2 7.25(s,4H)

(CDCl3)1.95(q,2H),2.45(t,2H),2.95(t,2H),3.65(s,3H), 5b C H NO 193.24 11 15 2 6.50-7.05(m,4H)

(CDCl3)1.50(d,9H),1.95(q,2H),2.48(t,2H),2.95(t,2H), 5b' C H NO 293.36 16 23 4 3.65(s,3H),6.98-7.40(m,4H)

(CDCl3)1.95(p,2H),2.45(t,2H),2.96(t,2H),3.65(s,3H), 5c C H NO S 225.31 11 15 2 3.72(broad,1H),6.62(d,2H,7.21(d,2H)

(CDCl3)1.51(d,9H),1.90(p,2H),2.42(t,2H),2.90(t,2H), 5c' C H NO S 325.42 16 23 4 3.66(s,3H),6.42(s,1H),7.32(s,4H)

(CDCl3)1.95(p,2H),2.45(t,2H),2.96(t,2H),3.65(s,3H), 5d C H FO S 225.31 11 13 2 6.96-7.44(m,4H)

5d' C11 H23 NO 4S 325.42 (CDCl3)1.51(d,9H),1.90(p,2H),2.42(t,2H),2.90(t,2H), 3.66(s,3H),6.42(s,1H),7.32(s,4H)

(CDCl3)1.83(p,2H),2.43(t,2H),2.82(t,2H),3.63(s,3H), 5e C H FO S 228.28 11 13 2 3.80(s,3H),6.82(d,2H),7.38(d,2H)

C H O S 240.32 (CDCl3)1.88(p,2H),2.45(t,2H),2.85(t,2H),3.64(s,3H), 5f 12 16 3 3.78(s,3H),6.82(d,2H),7.38(d,2H)

32 Table 2 Physical and Spectra Data of Compounds 1-9 (continued) Compound MF MW 1H NMR Data ( δ) (CDCl3)2.28(m,1H),2.40(m,1H),2.60(t,2H),3.64(s,3H), 6a C H Cl O 247.12 11 12 2 2 5.25(t,1H),7.20(m,4H)

(CDCl3)1.52(d,9H),2.40(m,2H),2.82(m,2H),3.64(s,3H), 6b C H ClNO 327.8 16 22 4 5.38(t,1H),7.18-59(m,4H)

(CDCl3)1.50(s,9H),2.32(m,2H),2.63(t,2H),3.69(s,3H), 6c C H ClNO 327.8 16 22 4 5.23(t,1H),6.60(s,1H),7.22-7.52(m,4H)

(CDCl3)2.35(m,2H),2.82(t,2H),3.70(s,3H),5.22(q,1H), 6d C H ClFO 230.66 11 12 2 7.00-7.60(m,4H)

(CDCl3)2.32(m,4H),3.70(s,3H),3.80(s,3H),5.10(q,1H), 6e C H ClO 242.7 12 15 3 6.90(d,2H),7.52(d,2H)

C H ClO S 274.76 (CDCl3)1.50(s,9H),2.33(m,2H),2.61(t,2H),3.70(s,3H), 6f 12 15 3 5.22(t,1H),6.60(s,1H),7.40(m,4H)

(CDCl3)0.95(m,6H),1.70(m,1H),1.82(m,2H),2.50(m,4H), 7a C H ClN O S 434.96 17 23 2 5 2 3.65(s,3H),4.10(t,1H),5.49(m,1H),7.40(m,4H)

(CDCl3)0.95(m,3H),1.40- 7a' C16 H21 ClN 2O5S2 420.93 1.90(m,4H),2.58(m,4H),3.83(m,5H), 4.05(m,1H),5.50(m,1H),5.75(s,1H),6.98-7.11(m,4H)

(CDCl3)0.95(m,3H),1.40- 7b C17 H25 N3O5S2 415.53 1.90(m,3H),2.58(m,4H),3.83(m,5H), 4.05(m,1H),4.98(s,1H),5.55(m,1H),6.98-7.11(m,4H)

(CDCl3)0.95(m,6H),1.40- 7b' C22 H33 N3O7S2 515.64 2.00(m,11H),2.50(m,4H),3.72(s,3H), 3.98(m,1H),5.41(m,1H),6.62-7.82(m,4H)

(CDCl3)0.95(m,6H),1.50(s,9H),1.65(m,3H), 2.50(m,4H), 7c' C H N O S 501.62 21 31 3 7 2 3.68(s,3H),3.95(m,1H),5.40(m,1H), 6.60-7.82(m,4H)

(CDCl3)0.95(p,3H),1.30- 7d C16 H21 FN 2O5S2 404.48 1.80(m,4H),2.50(m,4H),3.86(m,4H), 5.40(m,1H),6.98-7.60(m,4H)

(CDCl3)0.95(p,3H),1.30- 7e C17 H24 N2O6S2 416.51 1.80(m,4H),2.50(m,4H),3.63(s,3H), 3.80(s,3H),5.36(m,1H),6.83(m,2H),7,30-7.55(m,2H)

33 Table 2 Physical and Spectra Data of Compounds 1-9 (continued) Compound MF MW 1H NMR Data ( δ) (CDCl3)0.95(m,6H),1.70(m,1H),1.82(m,2H),2.50(m,4H), 8a C H ClN O S 448.98 18 25 2 5 2 2.80(d,3H),3.62(m,1H),3.65(s,3H),5.50(m,1H),7.40(m,4H)

(CDCl3)0.95(m,3H),1.20- 8a' C17 H23 ClN 2O5S2 434.96 2.00(m,4H),2.55(m,4H),2.80(s,3H), 3.62(s,3H),3.65(t,1H),5.57(m,1H),7.20(m,4H)

(CDCl3)0.95(m,6H),1.70(m,1H),2.56(m,4H),2.80(s,3H),

8b C18 H27 N3O5S2 429.55 3.63(s,3H),3.70(m,1H),5.50(m,1H),6.58(board,1H), 7.18-7.55(m,4H)

(CDCl3)0.95(m,6H),1.50(s,9H),1.65(m,3H),2.58(m,4H), 8b' C23 H35 N3O7S2 529.67 2.80(d,3H),3.62(s,3H),3.68(m,1H),5.50(t,1H),6.83- 7.60(m,4H)

(CDCl3)0.95(m,6H),1.05(m,4H),2.50(m,4H),2.80(s,3H), 8c C H N O S 415.53 17 25 3 5 2 3.65(m,4H),5.30(m,1H),6.60(d,2H),7.38(d,2H)

(CDCl3)0.95(m,3H),1.30- 8c' C22 H33 N3O7S2 515.64 1.99(m,13H),2.52(m,4H),2.80(s,1H), 3.65(m,4H),5.40(m,1H),6.62(s,1H),7.40(q,4H)

(CDCl3)0.95(p,3H),1.30- 8d C17 H23 FN 2O5S2 418.5 1.80(m,4H),2.50(m,4H),2.80(s,3H), 65(m,4H),5.45(m,1H),7.00(t,2H),7.60(m,2H)

(CDCl3)0.95(m,3H),1.30- 1.99(m,4H),2.50(m,4H),2.80(s,3H), 8e C H N O S 430.54 18 26 2 6 2 3.66(m,1H),3.68(s,3H),3.80(s,3H),5.39(m,1H),6.85(d,2H), 7.51(m,2H)

(CDCl3)0.95(m,3H),1.00-2.00(m,4H),2.40-2.60(m,11H), 9a C H N O S 515.6 21 29 3 8 2 3.63(m,4H),4.10(m,1H),5.40(m,1H),7.49(d,4H),8.09(s,1H)

(CDCl3)0.95(m,3H),1.00- 9b C25 H29 N3O8S2 563.64 2.00(m,4H),2.22(s,4H),2.55(m,4H), 2.80(m,3H),3.63(m,4H),5.40(m,1H),7.30-8.10(m,8H)

(CDCl3)0.95(m,3H),1.00- 2.00(m,11H),2.50(m,4H),3.50(q,2H), 9c C H N O S 533.7 26 35 3 5 2 2.80(m,5H),3.50(q,2H),3.62(s,3H),3.65(m,1H),4.92(s,1H), 5.39(m,1H),7.18-7.50(m,8H)

34

3.3 Biochemical Assays

Human neutrophil elastase. Incubation method. HNE was assayed by mixing 10 µL of 70 µM enzyme solution in 0.05 M sodium acetate/0.5 M NaCl buffer, pH 5.5, 10 µL dimethyl sulfoxide and 980 µL of 0.1 M HEPES buffer, pH 7.25, containing 0.5 M NaCl, in a thermostated cuvette. A 100 µL aliquot was transferred to an ermostated cuvette containing 880 µL 0.1 M HEPES/0.5 M NaCl buffer, pH 7.25, and 20 µL of a 70 µM solution of MeOsuc-Ala-Ala-Pro-Val p-NA, and the change in absorbance was monitored at 410 nm for 60 seconds. In a typical inhibition run, 10 µL of inhibitor (700 µM) in dimethyl sulfoxide was mixed with 10 µL of 70 µM enzyme solution and 980 µL 0.1 M

HEPES/0.5 M NaCl buffer, pH 7.25, and placed in a constant temperature bath. Aliquots

(100 µL) were withdrawn at different time intervals and transferred to a cuvette containing 20 µL of MeOSuc-Ala-Ala-Pro-Val p-nitroanilide (7 mM) and 880 µL 0.1 M

HEPES/0.5 M NaCl buffer. The absorbance was monitored at 410 nm for 60 seconds.

Progress curve method. Progress curves were generated by adding 5 µL of a 2.0 µM

HNE solution in 0.05 M sodium acetate buffer, pH 5.5, to 10 µL of inhibitor (50 µM solution in DMSO), 15 µL of substrate (MeOSuc-Ala-Ala-Pro-Val pNA, 7 mM in

DMSO) and 970 µL 0.1 M HEPES buffer/0.5 M NaCl buffer, pH 7.25, and the absorbance was monitored at 410 nm for ten minutes.

-1 -1 The apparent second-order inactivation rate constants (k inact /K I M s ) were determined in duplicate. Typical progress curves for the hydrolysis of MeO-Suc-AAPV p-NA by HNE in the presence of inhibitor (I) is shown in Figure 12. The release of p- nitroaniline was monitored continuously at 410 nm. The pseudo first-order rate constant

35 (k obs ) for the inhibition of HNE as a function of time was determined according to eq 1 below, where A is the absorbance at 410 nm, νo is the reaction velocity at t = 0, νt is the final steady state velocity, kobs is the observed first order rate constant to steady state, and A o is the absorbance at t = 0. The k obs values were obtained by fitting the A versus t data into eq 1 using nonlinear regression analysis (SigmaPlot, Jandel Scientific). The second order rate constants were determined by calculating kobs /[I] and then correcting for the substrate concentration using eq 2. Control curves in the absence of inhibitor were linear.

−kobst A = νst + {( νo − νs) (1 − e )} ⁄ k obs + Ao ------(1)

kinact ⁄K I= (kobs/[I]) (1 + [S]/ K m) ------(2)

36 CHAPTER 4 RESULTS AND DISSCUSION

4.1 Synthesis

Inhibitor (I) was readily synthesized using the reaction sequences outlined in Scheme

1. Briefly, the appropriate amino acid was converted into the corresponding methyl ester hydrochloride using thionyl chloride/methanol and the methyl ester was then reacted with the adduct of N-chlorosulfonyl isocyanate and t-butyl alcohol in the presence of TEA to give the amino acid-derived sulfamide derivative. This was readily deblocked using TFA and then cyclized with sodium hydride to give the heterocyclic intermediate 4. Reaction of 4 with an appropriate alpha-chlorothioether gave a diastereomeric mixture of 7. The desired alpha-chlorothioethers were readily obtained by alkylating a substituted thiophenol followed by alpha-chlorination with N-chlorosuccinimide. Intermediate 7 was alkylated with methyl iodide in the presence of sodium hydride to give (I). All reactions proceeded uneventfully in fair to good yields. The final products were obtained as mixtures of diastereomers which were then used in the biochemical studies without separating the two diastereomers.

37

Scheme 1 Synthesis of Inhibitors (I)

38

4.2 Biochemical Results

The inhibition activity of compounds 7a-9c toward HNE was then determined using incubation method. The K obs /[I] values, which reflect the potency of each inhibitor, are listed in Table 3.

Table 3 Biochemical Evaluation of Inhibitors (I)

HLE Compound R1 R2 R3 -1 -1 kobs /[I] (M s ) 7a n-Propyl methyl Cl 21 8e n-Propyl methyl F 326

8f n-Propyl methyl OCH 3 70

8d n-Propyl methyl p-NH 2 109 8d’ n-Propyl methyl p-NHBoc 166

9b n-Propyl methyl p-NHCO(CH 2)2COOH 297 9a n-Propyl methyl p-NHCOPhCOOH 263

9c n-Propyl methyl p-NHCONH(CH 2)2Ph 149 7b iso-butyl H Cl ND a 8b iso-butyl methyl Cl 1379 7c’ iso-butyl H m-NHBoc ND 8c’ iso-butyl methyl m-NHBoc ND 7d’ iso-butyl H m-NH 2 ND

8c iso-butyl methyl m-NH 2 ND ND a (not determined)

Incubation of inhibitor 8b with HNE led to rapid, time-dependent, irreversible loss of enzymatic activity (Figure 12). The k on and k off values of inhibitor 8b were 2606 M-1 s-1

-5 -1 and 4 × 10 s , respectively, yielding an apparent inhibition constant (K I) of 15 nM.

These values compare very favorably with “gold standard” inhibitors of HNE reported in the literature.31

39

Figure 12 Time-Dependent Loss of Enzymatic Activity (an inhibitor 8b to HNE ratio of 10 was used).

The bimolecular rate constant k inact /K I of inhibitor 8b , an index of inhibitor potency, was determined using the progress curve method 32 and found to be 4575 M -1 s -1 (Figure

13).

Figure 13 Progress Curves of Inhibitor 8b with HNE

40 CHAPTER 5 CONCLUSIONS

The design, synthesis and in vitro biochemical evaluation of a novel class of inhibitors of human neutrophil elastase (represented by structure (I)) has been described. Inhibitor

(I) is synthetically tractable and was designed to interact with the S1-Sn’ subsites of the enzyme. Since the S1 subsites of HNE and PR 3 are very similar (hydrophobic pockets of medium size) primary focus was placed on exploiting interactions with the S’ pockets.

The S’ subsites of HNE and PR 3 differ considerably; consequently optimal selectivity can be realized by exploiting these differences. As anticipated, the synthesized inhibitors inactivated HNE efficiently and in a time-dependent fashion. Furthermore, and although not established experimentally, inhibitor (I) is proposed to inactivate HNE via an enzyme-induced 3-Aza Grob fragmentation process by generating a highly reactive

Michael acceptor.

41

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42

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